The overall goal of this project is to gain a better understanding of the processes that underly and control ion transport in epithelial cells of Na transporting tissues. Reabsorption of ions in epithelial tissues such as the kidney and different parts of the gastrointestinal tract are of vital importance for the maintenance of proper composition and volume of the body fluids in health and disease. Of particular interest is the regulation of intracellular pH and its relation to the control of ion transport on the apical and basolateral sides of the epithelial cells since it is known that many intracellular processes and membrane transport mechanisms are highly pH dependent. We plan to determine whether regulation of apical Na conductance and of transepithelial Na and C1 transfer occurs in response to changes in intracellular pH that are induced by hormones or other experimental maneuvers. For these studies we will use preparations that serve as model transport systems, namely the frog skin with its principal cells (P-cells) and cultured toad kidney cells (A6 cells), a model for distal renal tubules. In the short-circuited frog skin, Na reabsorption proceeds via P-cells which form a functional syncytium. The action of hormones and the effect of other manuevers on intracellular pH, cell membrane potential, apical Na conductance will be measured with double barrel microelectrodes inserted into single epithelial cells while simultaneously determining Na reabsorption across the tissue with the short-circuit current. Of special interest are the effects of (1) hormones that are known to promote Na transport (antidiuretic hormone, aldosterone, insulin), (2) cAMP and agents that stimulate its production (forskolin) and inhibit its breakdown (phosphodiesterase inhibitors), and (3) of changes in Ca2+. In view of the hypothesis that changes of Na and Cl uptake into epithelial cells may be linked via changes in intracellular pH, active (i.e. transcellular) Cl transport and intracellular Cl activity will be measured under conditions in which changes in intracellular pH are observed. DIDS-inhibitable transepithelial Cl transport will be measured since Cl transfer on the apical side of the P-cells involves a DIDS-inhibitable C1/HCO3 exchange. In addition, the fluorescent dye BCECF will be used to examine the effects of insulin or aldosterone on intracellular pH in A6 cells while the short-circuit current is measured. The approaches delineated above have the decisive advantage that information on intracellular pH will be obtained in the same cell that is simultaneously monitored for hormone induced changes in transport.